The aim of this study was to show the reliability and validity of a Turkish version of Diabetes Eating Problem Survey-Revised (DEPS-R) in children and adolescents with type 1 diabetes mellitus.A total of 200 children and adolescents with type 1 diabetes, ages 9-18 years, completed the DEPS-R Turkish version. In addition to tests of validity, confirmatory factor analysis was conducted to investigate the factor structure of the 16-item Turkish version of DEPS-R.The Turkish version of DEPS-R demonstrated satisfactory Cronbach’s ∝ (0.847) and was significantly correlated with age , hemoglobin A1c levels , and body mass index-standard deviation score  indicating criterion validity. Median DEPS-R scores of Turkish version for the total samples, females, and males were 11.0, 11.5, and 10.5, respectively.Disturbed eating behaviors and insulin restriction were associated with poor metabolic control. A short, self-administered diabetes-specific screening tool for disordered eating behavior can be used routinely in the clinical care of adolescents with type 1 diabetes. The Turkish version of DEPS-R is a valid screening tool for disordered eating behaviors in type 1 diabetes and it is potentially important to early detect disordered eating behaviors. Noticing and treating eating disorders in children and adolescents with type 1 diabetes is important because of their potentially severe consequences. Using a screening tool designed specifically for individuals with type 1 diabetes when assessing disturbed eating behaviors in this population is important.No validated disease-specific short screening tool for children and adolescents with type 1 diabetes in Turkey has so far been established. The Turkish version of Diabetes Eating Problem Survey-Revised can be used as a valid screening tool for disordered eating behaviors in type 1 diabetes. This short, self-administered diabetes-specific screening tool for disordered eating behavior can be used in the clinical care of children and adolescents with type 1 diabetes.Adolescence is a developmental period with characteristic properties such as physiological changes  and psychological changes. These changes may lead to body image dissatisfaction and to an increase in prevalence of eating disorders, especially in females . The terEtiology of DEB is complex and multifactorial ,6. IndivEarly detection and treatment of eating disorders in children and adolescents with T1D is important because of their potentially severe consequences. Early detection is critical in this population in order to maintain optimum health status and decrease the chances of complications such as retinopathy, neuropathy, and diabetic ketoacidosis. The complexity of diabetes management in combination with eating disorder treatment, it is necessary to detect those most at risk as early as possible. A number of screening questionnaires and structured clinical interviews help to identify and diagnose eating disorders in children and young people with diabetes . It is iThe Diabetes Eating Problem Survey-Revised (DEPS-R) developed by Markowitz et al  is a diaIn this cross-sectional study, the children and adolescents were asked to answer a questionnaire during a regularly scheduled medical visit after written informed consent had been obtained from the subjects and their parents. All subjects were T1D patients who were being treated with multiple daily insulin injections or with insulin pump therapy. Patient records were reviewed for the following eligibility criteria:- Duration of type 1 diabetes ≥1 year,- A regular follow-up for at least 1 year,- No major medical problems .Two hundred adolescents with T1D-90 (45%) males, 110 (55%) females-aged 9–18 years were included in the study.DEPS-R is a 16-item diabetes-specific self-report questionnaire to test for diabetes-specific eating disorders. Answers are scored on a six-point Likert scale, with higher scores indicating more DEB and a total score of ≥20 indicating a high risk for eating disorders (range 0-80). The original DEPS-R has been shown to have a good internal consistency  and construct validity in a sample of pediatric population with T1D . Back-trThis study was approved by the Ege University Faculty of Medicine Clinical Research Ethics Committee . The purpose of the study was explained to each participant and written informed consent was obtained. The study procedures were in accordance with the Declaration of Helsinki.In all subjects, height was measured to the nearest centimeter using a rigid stadiometer. Weight was measured unclothed to the nearest 0.1 kg using a calibrated balance scale. Body mass index (BMI) was calculated by the weight (kg)/height (m²) equation. Standard deviation scores (SDS) for weight, height, and BMI were calculated according to age and gender using reference values for Turkish children and the participants were categorized into four different groups as underweight (BMI-SDS <-1), of normal weight (BMI-SDS ≥-1 - <+1), overweight (BMI-SDS ≥+1 - +2< BMI-SDS), and obese (BMI-SDS ≥+2) .Hemoglobin A1c (HbA1c) measurements were performed with capillary method using the NycoCard II Reader  device.All data are presented as median for skewed data , duration of T1D, and DEPS-R score), mean ± SD for BMI-SDS, or percent as indicated. Normal distribution was tested for continuous data. Group differences were investigated using the t-test for normally distributed data and the Mann-Whitney U test for data not showing a normal distribution. Correlations using Pearson’s and Spearman’s correlation coefficients were calculated to explore relationships of DEPS-R score with age, HbA1c (%), duration of T1D, and BMI-SDS. Correlation values of 0.10-0.29 were interpreted as small, 0.30-0.49 as medium, and 0.50-1.0 as large . The facInternal consistency: The Cronbach coefficients for the DEPS-R Turkish version were 0.847, 0.857, and 0.830 for the entire sample, females, and males, respectivelyConfirmatory factor analysis: After the suitability of data for factor analysis was assessed, confirmatory factor analysis was performed on the 16 items of the DEPS-R Turkish version. Among the main factors and sub-factors, the established model was statistically significant , 11.5 (0-55), and 10.5 (0-55), respectively. There was no significant difference between females and males (p=0.122). The median scores of the DEPS-R for the MDI group was 11.0 (0-47) and 11.0 (0-55) for the insulin pump group. There was no significant difference between MDI and insulin pump therapy groups (p=0.813). A recommended cut-off score of ≥20 has been empirically established as a threshold indicating the need for further clinical assessment of eating pathology .There were significant positive correlations of DEPS-R Turkish version scores with age , HbA1c levels , and BMI-SDS  among females. There was a positive small correlation between DEPS-R Turkish version scores and HbA1c levels  and there was positive medium correlation with BMI-SDS  among males .This study examined the psychometric properties of the Turkish version of DEPS-R and its clinical utility. The DEPS-R is a 16-item instrument designed to screen for DEB in T1D. Markowitz et al  first usThe confirmatory factor analysis is a kind of structural equation model which is used to determine the relationship between observable and latent variables and has a significant value in scale adaptation studies. After the suitability of data for factor analysis was assessed, confirmatory factor analysis was performed and confirmatory factor loadings of the subscales were shown to be at an acceptable level .In this sample, DEPS-R scores were similar to those reported in previous studies  . In addition, despite the fact that there were no significant differences in median DEPS-R scores 11.0 (0-55) between females and males in these studies, in our sample, DEPS-R scores of males were slightly higher than in previous studies .Previous studies defined a score of DEPS-R ≥20 as an indicator of high risk for DEB ,15,16. TConsistent with the literature, in our series also, patients with scores above the cut-off on DEPS-R had significantly higher HbA1c levels (p=0.002) in both genders ,15,20. N1c levels (10).First DEPS-R validation study showed positive correlations of DEPS-R scores with age, age- and gender-adjusted BMI and HbACriterion validity of the Turkish version of the DEPS-R was shown through significant positive medium correlations with BMI-SDS  and HbA1c levels  and a small but positive correlation with age  that may have been influenced by the presence of DEB in children and adolescents with T1D. Therefore, in clinical settings, we recommend assessment of DEPS-R score in relation to BMI-SDS and age in T1D patients. Higher BMI-SDS, higher HbA1c, and older age appear to be risk factors for the development of DEBs among children and adolescents with T1D.A major limitation of this study is its inability to validate the Turkish version of DEPS-R with a structured clinical diagnostic interview by a pediatric psychiatrist. This may have led to overestimation of DEB.Our data has shown that children and adolescents with T1D who have higher BMI values and higher HbA1c levels appear to exhibit more DEBs. DEB and insulin restriction were associated with poor metabolic control. Diabetes health care professionals should be aware of comorbid eating disorders in children and adolescent with T1D. A short, self-administered diabetes-specific screening tool for DEB can be used routinely in the clinical care of children and adolescents with T1D. The Turkish version of DEPS-R is a valid screening tool for DEB in T1D and it is potentially important to detect DEB at an early stage. We propose that future research should focus on the validity of the DEPS-R as compared with a structured clinical diagnostic interview.


Feasible peripheral biomarker for Alzheimer’s disease (AD) is lacking. Dysregulation of N-methyl-D-aspartate (NMDA) receptor is implicated in the pathogenesis of AD. D-amino acid oxidase (DAO) and amino acids can regulate the NMDA receptor function. This study aimed to examine whether peripheral DAO and amino acids levels are characteristic of age-related cognitive decline. We enrolled 397 individuals (including amnestic mild cognitive impairment (MCI), mild AD, moderate to severe AD, and healthy elderly). DAO levels in the serum were measured using ELISA. Amino acids levels in serum were measured by high performance liquid chromatography. Severity of the cognitive deficits in subjects was assessed using Clinical Dementia Rating Scale (CDR). The DAO levels increased with the severity of the cognitive deficits. DAO levels were significantly associated with D-glutamate and D-serine levels. The Receiver Operating Characteristics analysis of DAO levels for AD patients vs. healthy controls determined the optimal cutoff value, 30.10, with high sensitivity (0.842) and specificity (0.889) (area under curve = 0.928). This is the first study indicating that the peripheral DAO levels may increase with age-related cognitive decline. The finding supports the hypofunction of NMDA receptor hypothesis in AD. Whether DAO could serve as a potential surrogate biomarker needs further studies. Therefore, early detection and treatment can help to prevent or slow the progression of AD.Dementia is gaining increasing attention because of the high morbidity and mortality it causes in the population. Although cognitive deterioration is common in old age, the relationship between aging and degenerative dementia such as Alzheimer’s disease (AD) remains unclear. Whereas aging is a risk factor for AD, AD may not be an inevitable part of the aging process. The pathological changes that are detected in the brains of patients with AD, such as the presence of amyloid plaques and neurofibrillary tangles, are considered to appear several years before the development of clinical symptoms2, and its signaling also underlies complex neuronal activities such as learning and memory3. The N-methyl-D-aspartate (NMDA) receptor, a subtype of ionotropic glutamate receptor, plays an important role in synaptic plasticity, learning, memory, and cognition4. In AD patients, glutamate levels were diminished in the cerebrospinal fluid (CSF)5 and in ante mortem brain tissue6, the number of glutamate terminals in the hippocampus were decreased7, and low levels of D-serine (an endogenous full agonist of the glycine site of NMDA receptor) and high levels of L-serine were observed in the serum8. Another study found that D-serine levels were higher in the CSF of probable AD patients than in control subjects9. The density of NMDA receptor also decreases with age10. Therefore, dysfunction in the glutamate neurotransmission via NMDA receptor may contribute substantially to the pathophysiology of AD.Several mechanisms have been reported to be implicated in the pathogenesis of AD, one of them is dysregulation of glutamate neurotransmission. Glutamate plays a critical role in regulating neurogenesis, neurite outgrowth and synaptogenesis, neuronal survival, and synaptic plasticity11 relies mainly on clinical manifestations. Favorable laboratory tests, particularly from peripheral approach, are still lacking. Gene expressions associated with modulating NMDA receptor function may be involved in the etiology of MCI and AD. D-amino acid oxidase (DAO) is a flavoenzyme that degrades D-amino acids, mainly D-serine14. Studies indicate that aging is related with reduced D-serine levels and D-serine treatment decreases the extent of neuron death, suggesting that D-serine has neuroprotective effect against apoptosis15. D-serine is critical for the proliferation and neuronal differentiation of neural stem cells16. A body of evidence suggests that DAO also plays a key role in the process of oxidative stress18.To date, the diagnosing of AD or mild cognitive impairment 19. The aforementioned evidences suggest that DAO and its regulatory function on NMDA receptor may play important roles in the process of aging and its related cognitive decline.Inhibiting the activity of DAO is one of the avenues to enhance NMDA activation. Our previous study demonstrated that sodium benzoate, a DAO inhibitor, is better than placebo in improving the cognitive function of patients with MCI or mild AD in a randomized, double-blind, placebo-controlled trial20. Peripheral gene expression may be a useful surrogate for gene expression in the CNS when the relevant gene is expressed in both21. We hypothesize that the level of DAO and related amino acids  in the peripheral blood may be associated with age-related cognitive decline. This study aims to examine whether DAO was over-expressed in people with age-related cognitive deficits, and could serve as a surrogate diagnostic biomarker.It is difficult to collect sample from brain tissue. Thus, developing accessible peripheral biomarkers becomes more important for mental illnessA total of 397 subjects were enrolled: 116 healthy controls, 77 patients with amnestic MCI, 128 patients with mild AD, and 76 patients with moderate to severe AD. There were significantly more male subjects in the healthy control group than the other three groups (p = 0.027). There were significant differences in age distribution and educational levels among the four groups (p < 0.001). The percentage of subjects using anti-dementia agents (including AChEI and memantine) were also significantly different among the amnestic MCI, mild AD, and moderate-severe AD groups (p = 0.001). Among the amino acids that we measured, there were significant inter-groups differences for L-glutamate level, D-glutamate level, L-serine level, D-serine level and D to L-glutamate ratio . The demographic data of the four groups are summarized in Table We further matched the four groups in terms of gender, age, and education. In the matched cohort, there were no significant difference among the four groups in gender, age, and education . Among the amnestic MCI, mild AD, and moderate-severe AD groups, there was also no significant difference in the frequence of anti-dementia agents use p = 0.072). Among the amino acids that we measured, tIhere were significant inter-groups differences for D-glutamate level, glycine level, and D to L-glutamate ratio  in the matched cohort. The demographic data of the matched cohort are summarized in Table . Among tThe DAO levels in the healthy controls, amnestic MCI, mild AD, and moderate to severe AD were 23.9 ± 11.2, 32.2 ± 10.8, 38.1 ± 14.4 and 41.4 ± 19.5, respectively (p < 0.001)  . In the overall cohort, DAO level was significantly associated with D-glutamate level, D-serine level and L-alanine level (adjusted R square = 0.290) Table . In the Multiple linear regression analyses were used to test the potential factors associated with CDR score. The regression models were adjusted with age, sex and education. Similarly, D- to L- forms amino acids ratios were not entered in the regression model due to high co-linearity with amino acids levels. In the overall cohort, CDR score was significantly associated with age, education, DAO level, D-glutamate level and D-serine level (adjusted R square = 0.420)   vs. the proportion of false-positive results (1 - specificity). Patients with amnestic MCI were excluded for this analysis because amnestic MCI has not yet been confirmed as a definite diagnosis. In the overall cohort, the ROC analysis revealed that DAO level for all AD patients vs. healthy controls determined an optimal cutoff value, 29.74, with a good sensitivity (0.823) and modest specificity (0.767) (AUC = 0.868). D-glutamate had the highest AUC (0.798) among all amino acids with an excellent sensitivity (0.967) and modest specificity 0.531) at the optimal cutoff value 975.04. D/L-glutamate ratio had similar AUC (0.784) with a good sensitivity (0.833) and modest specificity (0.708) at the optimal cutoff value 0.18. D-serine level also had good AUC (0.740) with a modest sensitivity (0.635) and good specificity (0.800) at the optimal cutoff value 35.85. We further combined DAO and amino acid levels to generate an equation by logistic regression model. The combination of DAO, D-glutamate and D-serine had good AUC (0.832) with a modest sensitivity (0.645) and excellent specificity (0.967) at the optimal cutoff value 37.00  and good specificity (0.889) (AUC = 0.928). D/L-glutamate ratio had the highest AUC (0.834) among all amino acids with a good sensitivity (0.889) and modest specificity (0.737) at the optimal cutoff value 0.18. D-glutamate level had similar AUC (0.813) with a good sensitivity (0.889) and modest specificity (0.632) at the optimal cutoff value 1054.24. D-serine level also had good AUC (0.739) with a modest sensitivity (0.544) and good specificity (0.833) at the optimal cutoff value 39.19. We also combined DAO and amino acid levels to generate an equation by logistic regression model. The combination of DAO and D-serine had excellent AUC (0.940) with a modest sensitivity (0.783) and excellent specificity (1.000) at the optimal cutoff value 80.18 22. An earlier study found that D-serine level declines and DAO increases in the cerebellum of rats during early postnatal development23. A recent study found that the DAO mRNA expression levels were higher in the cerebellum compared to other brain regions, and the DAO mRNA levels were positively correlated with age less than 2 years in the cerebellum and amygdala in normal human post-mortem brain samples24. The above in vitro and in vivo studies suggest that neurotransmission via NMDA receptor is pivotal for regulating the cognitive function in the aging brain.The findings from peripheral blood in our study support the previous study that D-serine levels were higher in the CSF of AD patients than in control subjects25, saliva26 and urine27. D-glutamate has been detected in several brain areas, peripheral tissues, plasma and urine of rodents by Hamase’s group29. The D-glutamate level and other amino acid levels in this study were similar to those in a previous study in human plasma30. Mangas et al. found that the D-glutamate levels were increased in several brain regions that are important for cognitive and behavioral regulations in rats31. Although L-glutamate is the most abundant excitatory neurotransmitter in the brain32, D-glutamate level is relatively higher in the cortex than other brain regions33, suggesting that D-glutamate may be important for high cortical functions. It is noteworthy to investigate the role of D-glutamate in aging in future studies.D-glutamate level was found to decrease with the severity of cognitive impairment in this study. D-glutamate can be detected in bloodOur finding of increased DAO and D-serine levels as well as decreased D-glutamate levels for people with cognitive aging in the matched cohort were very similar to that of the overall cohort. The findings suggest that the changes of DAO, D-glutamate and D-serine with cognitive deterioration are not affected by the demographic characterics including age, sex and education. This observation supports the viewpoint that cognitive decline may be not necessarily an inevitable part of the normal aging process. However, whether DAO and amino acids levels in patients with cognitive impairment really progress with aging needs further elucidation by prospective study.DAO gene from the general population34, the peripheral blood-CNS relationship of DAO and amino acids expressions also needs to be examined by studies among individuals with AD. Third, the amino acids levels were not measured for all participants in this study due to limited amounts of blood samples. However, the average sample size with amino acids levels in each group for the matched cohort is around 22, making the results still of some value. Fourth, our findings need to be testified in various racial populations by other groups. Fifth, D-aspartate level was not assayed in this study. D-aspartate can bind to the glutamate site of GluN2 receptor to enhance the NMDA receptor function35. Animal studies found that D-aspartate enhanced NMDA receptor-dependent long-term potentiation in the hippocampus36, and rescued the synaptic plasticity decay in the hippocampus of aged mice35. The role of D-aspartate in cognitive aging deserves further investigation. Lastly, other neurodegenerative diseases  that are associated with altered DAO level38 could not be entirely ruled out although we had excluded subjects with obvious brain or mental disorders by history, physical examinations and laboratory assessments.Our study has several limitations. First, since this is a cross-sectional study, whether the findings keep constant over cognitive deteriorating process requires further prospective, longitudinal study. Second, although a previous GWAS study has found a suggestive evidence of association for the D-serine plasma-CSF ratio at the 39. Since AD is a complex and multifactorial disease, it is reasonable to combine different potential biomarkers for assisting the diagnosis. For example, combining DAO level with other potential tools, e.g. amino acids levels and β-amyloid40, for ascertainment might be a possible approach. In the future, the potential relationship of DAO levels and amino acids levels with treatment response for AD or a subpopulation of AD) requires clarification from larger samples of patients with different severities of cognitive deficits and under various treatments.Taken together, peripheral DAO levels are higher in patients with cognitive decline. The findings support the hypofunction of NMDA receptor-mediated neurotransmission hypothesis in MCI and ADAll subjects were screened and recruited from the following institutes: Department of Psychiatry of Kaohsiung Chang Gung Memorial Hospital, which is a major medical center in southern Taiwan, Department of Psychiatry of China Medical University Hospital, which is a major medical center in central Taiwan, and Department of Psychiatry of Taipei City Municipal Hospital, which is a major medical center in northern Taiwan. This study was approved by the institutional review boards of the aforementioned institutes, and carried out in accordance with the Declaration of Helsinki. After thorough description of the study to the subjects, written informed consents were obtained according to the IRB’s guidelines.41 criteria for probable AD and had a Clinical Dementia Rating (CDR)42 score of 1, or criteria for amnestic MCI43 of a presumably degenerative nature defined as subjective memory complaint corroborated by an informant and insufficient global cognitive and functional impairment to meet NINCDS-ADRDA criteria and had a CDR score of 0.5, [2] had sufficient education to communicate effectively and were capable of completing the assessments of the study, and [3] agreed to participate in the study and provided informed consent. Exclusion criteria included history of significant cerebrovascular disease; Hachinski Ischemic Score >4; major neurological, psychiatric or medical conditions other than AD; substance  abuse or dependence; delusion, hallucination or delirium symptoms; severe visual or hearing loss; and inability to follow protocol.All subjects were Han Chinese, aged 50–100 years, who were physically healthy and had normal laboratory assessments . Both patients and healthy controls were evaluated by research psychiatrists after a thorough medical workup. Patients were enrolled into this study if they [1] satisfied NINCDS-ADRDA There was no any Axis I or II psychiatric disorder for the healthy volunteers. All participants had no DSM-IV diagnosis of substance  abuse or dependence to exclude potential confounding effects. Patients with amnestic MCI had a CDR score of 0.5. Patients with mild AD had a CDR score of 1 and patients with moderate to severe AD had a CDR score 2 or greater.Both anti-dementia drugs free and medicated patients with AD were recruited for examining possible drug effects on the DAO level. Drug-free AD patients had not taken any anti-dementia drug for at least three months. For patients who had already been on anti-dementia drugs therapy, anti-dementia drugs had to be continued for at least three months with unchanged dose before enrollment. Psychotropic use history was ascertained by interviewing the patients and their family members or care givers, contacting other health care providers and reviewing medical records. Healthy individuals had no history of exposure to anti-dementia drugs. All patients with AD were recruited from the outpatient clinic at the aforementioned institutes, and all healthy volunteers were from the communities in northern, central and southern Taiwan.Among 281 patients with amnestic MCI or AD, 216 were anti-dementia drug free for 3 months or longer and the other 65 were stabilized on anti-dementia drugs  for at least 3 months. Among the drug-free patients, 206 patients were drug-naïve and the other 10 were nondrug-naïve.44. MMSE is a commonly used tool for the measurement of cognitive impairment and the screening for dementia in the elderly people44. However, one of the disadvantages to the utilization of the MMSE is that it is affected by age and education45. Another disadvantage of the MMSE is its lower level of sensitivity for mild degrees of impairment46.The participant’s cognitive function was assessed by CDR and Mini-Mental State Examination (MMSE)47. Besides, studies have shown that the CDR appears to be a reliable and valid tool for assessing and staging dementia with moderate to high overall inter-rater reliability48. Therefore, the grouping of subjects and further analysis with blood parameters in this study were based on CDR scores, which can reflect the global cognitive impairment of the subjects and not be affected by age and education level49.On the other hand, CDR exhibits excellent discriminatory ability in the very mild stages of dementiaFor both patients and healthy controls, blood sampling was done during 8–12AM after fasting for more than eight hours. Ten ml of blood was collected by personnel trained in phlebotomy using sterile technique. The blood specimens were processed immediately by centrifugation at 1000 x g. After centrifugation, serum was quickly dissected, immediately stored at −80 °C until further measurement.DAO protein concentrations were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits according to the manufacture’s recommended protocol . Briefly, 100 μL serum samples and the standard were added to each well of a 96-well plate. The solutions were incubated for 1 hour at 37 °C. The liquid was then removed. 100 μL Detection Reagent A was added to each well and incubated for 1 hour at 37 °C. Each well was washed for 3 times. 100 μL of Detection Reagent B was added to each well, and the solutions were incubated for 30 minutes at 37 °C. Each well was washed for 5 times and then incubated with 90 μL substrate solution for 20 min at 37 °C with the protection from light. 50 μL stop solution was added to each well. A Benchmark Plus Microplate Reader (Bio-Rad) was used to read the optical density at 450 nm. The concentrations of DAO in the samples were determined according to a standard curve.According the instruction manual of the DAO ELISA kit, the assay has high sensitivity  and excellent specificity for detection of DAO. To the best of current skills and knowledge, no significant cross-reactivity or interference between DAO and analogues was observed.Serum was firstly extracted by methanol , then filtered after 15 min centrifugation (1500 × g) with nylon membranes . The filtrate was diluted with proper amount of 20% methanol then derivatized with N-isobutyl-L-cycteine (IBC) and O-phthaldialdehyde mixture for 5 minutes then injected into high performance liquid chromatography  for analysis. Analytical column  with guard column  were used for the determination. Isocratic elution of mobile phase A  and B (50 mL acetonitrile in 600 mL methanol) were performed under fluorescence detection , respectively. Retention time of each amino acid was L-glutamine, 25.5 min; D-glutamine, 27.3 min; L-serine, 33.6 min; D-serine, 35.8 min; glycine, 41.5 min; L-alanine, 47.2 min; D-alanine, 50.3 min, respectively. All amino acids levels were double-checked by performing HPLC analyses for two times in order to confirm that the peaks were not artifact.All subjects’ clinical characteristics, DAO levels and amino acids levels were presented as mean ± SD or number (percentage). All statistical methods were performed using IBM SPSS Statistics version 22.0 (SPSS inc.). All mean values between groups were compared using independent t test or Mann-Whitney U test for two groups, one-way ANOVA or Kruskal-Wallis test for three groups, and percentages using χ2 test. Multiple linear regression and Receiver Operating Characteristics (ROC) analysis were used to generate predictive models and to evaluate for the significant predictors of AD patients. A P value less than 0.05 was considered statistically significant.Supplementary Table 1


Because etomidate induces prolonged adrenal suppression, even following a single bolus, its use as an infused anesthetic is limited. Our previous study indicated that a single administration of the novel etomidate analog methoxyethyletomidate hydrochloride (ET-26-HCl) shows little suppression of adrenocortical function. The aims of the present study were to (1) determine the minimum infusion rate of ET-26-HCl and compare it with those for etomidate and cyclopropyl-methoxycarbonylmetomidate (CPMM), a rapidly metabolized etomidate analog that is currently in clinical trials and (2) to evaluate adrenocortical function after a continuous infusion of ET-26-HCl as part of a broader study investigating whether this etomidate analog is suitable for long infusion in the maintenance of anesthesia.n = 32) were then randomly divided into four groups: etomidate, ET-26-HCl, CPMM, and vehicle control. Rats in each group were infused for 60 min with one of the drugs at its predetermined minimum infusion rate. Blood samples were drawn initially and then every 30 min after drug infusion to determine the adrenocorticotropic hormone-stimulated concentration of serum corticosterone as a measure of adrenocortical function.The up-and-down method was used to determine the minimum infusion rates for ET-26-HCl, etomidate and CPMM. Sprague-Dawley rats ; however, this reduction did not reach statistical significance. Thus, further studies are warranted examining the practicability of using ET-26-HCl as an infused anesthetic. Etomidate, one of the most frequently used intravenous anesthetics, has many favorable properties, such as its ability to maintain hemodynamic stability and generate low incidences of respiratory depression and anaphylaxis. However, the adrenocortical insufficiency caused by etomidate restricts its clinical applications. Several studies have shown that as little as a single administration of etomidate may induce adrenocortical insufficiency, and the increased propensity for this may last 48 h after the administration of etomidate .In the 1980s, etomidate was infused in a continuous manner in critical patients to maintain sedation; however, studies later showed that this dosage regimen increased mortality . PharmacGuide to the Care and Use of Experimental Animals . Five animals per cage were housed under standard conditions at a temperature of 22 °C and a humidity of 60% and with standard laboratory rat chow and water. The animals were allowed to acclimatize for 1 week.All animal protocols used in the present study were approved by the Ethics Committee of the West China Hospital, Sichuan University, China . Sprague Dawley rats weighing 225–350 grams were purchased from Chengdu Dassy Biological Technology Co. Ltd.  and cared for in accordance with the the Canadian Council on Animal Care’s Etomidate (2 mg/mL) formulated as an emulsion was purchased from B. Braun Melsungen AG, and propofol (10 mg/mL) formulated as an emulsion was purchased from AstraZeneca. The Laboratory of Anesthesia and Critical Care Medicine  synthesized ET-26-HCl using a previously published approach; ET-26-HCl was formulated as an aqueous solution (10 mg/mL) with 35% propylene glycol and then diluted with normal saline  to 6 mg/mL. Cyclopropyl-methoxycarbonylmetomidate (CPMM) was also synthesized by our laboratory according to the issued patent  and formulated as an aqueous solution (8 mg/mL) with 20% sulfobutylether-β-cyclodextrin .An infusion pump  was used to administer the sedatives and hypnotics, and a homoeothermic blanket was used to maintain the body temperature of the rats at 36–38 °C while they were anesthetized. A pulse oximeter placed on the upper right hind leg was used for monitoring oxygen saturation. Heart rate and rhythm and the respiratory rate were monitored with a BL-420S biological data acquisition and analysis system . Oxygen (100%) was delivered at a rate of 2 L/min to each anesthetized rat through a face mask connected to a coaxial circuit.n = 60) were randomly assigned to three groups that received either etomidate, ET-26-HCl, or CPMM.Rats  for each anesthetic was determined as previously described . The inin = 32) were then randomly divided into the following four groups, with eight rats in each group: etomidate, ET-26-HCl, CPMM, or propylene glycol vehicle control, and the infusion protocol started (see g) for 5 min. The supernatant was transferred to a clean Eppendorf centrifuge tube and subjected to a second centrifugation . After the second centrifugation, the supernatant was removed again, and a final high-speed centrifugation was performed to remove all red blood cells and particulate. The serum was transferred to a clean tube and immediately placed at −20 °C. The corticosterone concentration in each serum sample obtained at the various time points was quantified within 1–2 days using an enzyme-linked immunosorbent assay  and a 96-well plate reader.The infusion protocol for each rat began at 8:30–9:00 a.m. to minimize natural changes in corticosterone levels. After the rats were weighed and the caudal vein was catheterized, dexamethasone (0.2 mg/kg) was administered. Two hours later, the baseline blood sample was drawn. Rats . Figure preparation and curve fitting was performed with Prism version 5.0 .All data are presented as the mean ± standard deviation. For comparisons of serum corticosterone concentrations after infusion of vehicle and test compounds, a one-way analysis of variance was conducted followed by Tukey’s honest significant difference test or the Tamhane test. A value of The MIRs of etomidate, CPMM, and ET-26-HCl were 0.29, 0.95, and 0.62 mg/kg/min, respectively .The baseline concentrations of serum corticosterone (before the start of the drug infusion) were not significantly different among the four groups and averaged 185.86 ± 68.66 ng/mL. However, serum corticosterone concentrations increased over time in all groups after drug infusion and ACTH stimulation. Compared with the control group, rats administered etomidate demonstrated significantly lower corticosterone concentrations at 60 min, which is the end of the drug infusion, as well as 90, 120, 150, and 240 min after the beginning of drug infusion. Compared with the etomidate group, rats administered ET-26-HCl showed significant differences 90, 120, 180, 210, and 240 min after begin of drug infusion. Compared with rats administered CPMM, rats administered etomidate showed significant differences in serum corticosterone concentrations at the end of the drug infusion as well as 60, 90, 180, and 210 min after begin of drug infusion. ACTH-stimulated serum corticosterone concentrations in rats administered CPMM or ET-26-HCl were not significantly different from each other at any time .in vivo plasma concentration of drugs continuously infused at a constant rate reaches equilibrium at 4–5 half-lives. We determined that the MIR for etomidate was 0.285 mg/kg/min, for ET-26-HCl it was 0.62 mg/kg/min, and for CPMM it was 0.95 mg/kg/min. These results suggested that the anesthetic efficacy of ET-26-HCl was approximately one-half to one-third of that for etomidate, which is consistent with the results of our previous study (50 (effective dose for 50 percent of the group) of CPMM is 0.89 ± 0.18 mg/kg/min  or no (−) reaction to a painful stimulus, an up-and-down design method previously described . Each hyus study . In addig/kg/min . These aAfter determining the MIRs, we next examined the effects of etomidate, ET-26-HCl, and CPMM continuously infused for 1 h and inducing the same anesthesia depth on serum corticosterone concentrations. Compared with those in control rats, ACTH-stimulated serum corticosterone concentrations were significantly decreased by etomidate, while those following ET-26-HCl or CPMM administration were not associated with a significant difference. When ACTH-stimulated serum corticosterone concentrations in rats administered etomidate were compared with those in rats infused with vehicle, ET-26-HCl or CPMM, all time points examined after the drug infusions, except 180 min and 240 min, show significant differences with etomidate. The corticosterone concentrations tended to be reduced for the first hour following ET-26-HCl infusion (as compared to vehicle infusion); however, this reduction did not reach statistical significance. We concluded that ET-26-HCl does not induce obvious inhibition of adrenal function.The safe dosage range of etomidate has been diminishing owing to its inhibition of adrenocortical function, as the suppression following even a single bolus may last 72 h . This inThe method to evaluate minimum infusion rate of present study is feasible, and it could maintain a similar anesthesia depth of all drugs. The corticosterone concentrations tended to be reduced for the first hour following ET-26-HCl infusion (as compared to vehicle infusion) was mainly because of the molecular structure of ET-26-HCl still have some depression to the release of corticosterone; however, this reduction did not reach statistical significance. Thus, further studies are warranted examining the practicability of using ET-26-HCl as an infused anesthetic.10.7717/peerj.3693/supp-1Data S1Click here for additional data file.


This study aimed to clone the NKA α-subunit (NKAα) from the inner mantle adjacent to the extrapallial fluid of Tridacna squamosa, to determine its subcellular localization, and to examine the effects of light exposure on its transcript level and protein abundance. The cDNA coding sequence of NKAα from T. squamosa comprised 3105 bp, encoding 1034 amino acids with an estimated molecular mass of 114 kDa. NKAα had a basolateral localization along the shell-facing epithelium of the inner mantle. Exposure to 12 h of light led to a significantly stronger basolateral NKAα-immunofluorescence at the shell-facing epithelium, indicating that NKA might play a role in light-enhanced calcification in T. squamosa. After 3 h of light exposure, the transcript level of NKAα decreased transiently in the inner mantle, but returned to the control level thereafter. In comparison, the protein abundance of NKAα remained unchanged at hour 3, but became significantly higher than the control after 12 h of light exposure. Hence, the expression of NKAα in the inner mantle of T. squamosa was light-dependent. It is probable that a higher expression level of NKA was needed in the shell-facing epithelial cells of the inner mantle to cope with a rise in Na+ influx, possibly caused by increases in activities of some Na+-dependent ion transporters/channels involved in light-enhanced calcification.Na Symbiodinium) which are found extracellularly in a branched tubular system embedded in their tissues. With the help of symbiotic zooxanthellae, giant clams can grow at high rates in nutrient deficient tropical waters, but the availability of light critically affects their growth and rate of shell formation . It comprised 3105 bp, encoding 1034 amino acids with an estimated molecular mass of 114.6 kDa , but different from those of Nkaα1a (the freshwater-isoform) and Nkaα1b (the seawater-isoform) of A. testudineus  than the latter .In antibody . The speantibody . Unlike antibody , the seaantibody . The NKAantibody  of clamsantibody . Indeed,NKAα in the inner mantle of T. squamosa exposed to light for 3 h was significantly lower than that of the control, but it returned back to the control level at 6 h and 12 h of light exposure (P<0.05) protein abundance of NKAα in the inner mantle of T. squamosa after 12 h of exposure to light as compared with the control , NKA is expressed in the basolateral membrane of calicoblastic cells [Galaxea fascicularis) is inhibited by the Ca2+-ATPase inhibitor, ruthenium red, and by the NKA inhibitor, ouabain, indicating the involvement of active Ca2+ transport and Na+/Ca2+ exchange in the process [G. fascicularis. As for T. squamosa, we had obtained the full coding sequence of a NKAα from its inner mantle, which was localized predominantly to the shell-facing epithelium. We had also demonstrated that exposure to light upregulated the protein abundance of NKAα in the inner mantle and increased the anti-NKAα immunofluorescence of the shell-facing epithelium, denoting the possibility of its participation in light-enhanced calcification.There is indirect evidence which suggests the involvement of NKA in biomineralization and calcification in animals. Before molting in terrestrial isopods, a large quantity of CaCO of Ca2+ . The aviormation . For coric cells . Calcifi process . Specifi process . Hence, + and two K+ binding sites, as well as essential residues in the transmembrane domains, which coordinate the binding sites to release one type of cation and then bind to the other [T. squamosa. Through mutational studies, Asp-742 in pig NKAα1 (corresponding to Asp-758 in T. squamosa) was shown to be a cytoplasmic K+ site [+ site was particularly important as it has been shown to stimulate the translocation of Na+ to the exterior [+ binding, and Na+ affinity is reduced by 96% when this motif was deleted from the C-terminus of NKAα [T. squamosa, Hydra vulgaris, and Schistosoma mansoni, the first amino acid is lysine (i.e. KETYY). In Crassostrea gigas and Ascaris suum, the lysine residue is substituted by arginine (i.e. RETYY), while in Doryteuthis opalescens, it is replaced with asparagine (i.e. NETYY). This might indicate a variation in Na+ affinity among NKAα of mollusks, as demonstrated among T. squamosa, C. gigas, and D. opalescens.NKAα comprises three Nahe other . A compahe other  reveals  K+ site  and thisexterior –32. The  of NKAα . AlthougT. squamosa. Two other motifs, Thr-Gly-Asp (TGD) and Thr-Gly-Asp-Gly-X-Asn-Asp (TGDGXND) are also conserved in NKAα of these invertebrates. They are involved in Mg2+ coordination associated with ATP binding at the phosphorylation site [Bufo marinus [T. squamosa (Ser-954). Site-directed mutagenesis of Nkaα from B. marinus indicates Thr-10 and Ser-11 as the cAMP-dependent PKC phosphorylation sites [H. vulgaris, S. mansoni, and A. suum lack these PKC phosphorylation sites, but Thr-23 (corresponding to Thr-10 of B. marinus) is present in the NKAα of T. squamosa.All P-type ATPases including NKA contain the Asp-Lys-Thr-Gly-Thr (DKTGT) motif, of which the aspartate residue represents a phosphorylation site. This motif is conserved in NKAα of all the invertebrates examined, inclusive of ion site . NKAα caion site . Ser-944 marinus , 37. Thion sites . The NKAT. squamosa, express only one form of NKAα. By contrast, multiple Nkaα isoforms have been identified in fish gills. For instance, the gills of the climbing perch, A. testudineus, typically express Nkaα1a, Nkaα1b, and Nkaα1c when exposed to fresh water, seawater and ammonia (in fresh water), respectively [+ and K+ binding [A. testudineus [T. squamosa consists of Asn-794  which its gills . In addihan NH4+ . Notablytudineus . As expehan NH4+ . Incidenand NH4+ . When NHactivity . Hence, +-ATPase ,43, but squamosa .T. squamosa to light leads to a significant increase in the Na+/NH4+-activated-NKA activity in the inner mantle, attributable to an increase in the effectiveness of NH4+ to replace K+ for NKA activation [T. squamosa after 12 h of light exposure. However, they do not offer a satisfactory explanation on why a change in the affinity of NKA to K+ and NH4+ would occur. This is because, similar to many other invertebrates, the inner mantle of T. squamosa expressed only one form of NKAα. It has been proposed previously [+ and NH4+ in the inner mantle of T. squamosa could result from changes in expression of NKAβ, because NKAβ is known to alter the activity of NKA by modulating the affinity of NKAα for Na+ and K+ [NKAβ/NKAβ expression and its interaction with NKAα in the inner mantle of T. squamosa.Exposure of tivation . Indeed,eviously  that cha+ and K+ . TherefoT. squamosa. This asymmetric distribution of NKA in epithelial cells facilitates and determines the vectorial transepithelial transport of water and certain ions across the epithelium [T. squamosa. However, with its basolateral localization, it is unlikely to be involved in the uptake of H+ as NH4+ in the extrapallial fluid by the shell-facing epithelial cells as suggested previously [In agreement with immunocytochemical and histochemical localization of basolateral NKA in non-excitable epithelial tissues of insects , 46, cruithelium . Notablyeviously , and theNKAα in the inner mantle of T. squamosa, but the protein abundance of NKAα remained unchanged as compared to the control in darkness. In fact, the NKAα protein abundance increased progressively from hour 3 onwards and became significantly greater than the control value at the 12th h of light exposure. The reason for the transient decrease in NKAα transcript level is unclear at present, but it would have minimal physiological significance due to the unchanged NKAα protein abundance. The significant increase in protein abundance of NKAα after 12 h of light exposure could be related to an upregulation of its production through increased translation or a downregulation of its degradation. Either way, it can be concluded that NKAα, and hence NKA activity, was regulated mainly at the protein level. A role for the NKA in light-enhanced calcification in T. squamosa is further supported by a significantly stronger basolateral NKAα-immunofluoresence of the shell-facing epithelium of clams exposed to light for 12 h. It is probable that more NKAα was needed in the shell-facing epithelial cells of the inner mantle to balance a rise in Na+ influx, possibly caused by increases in activities of some Na+-dependent ion transporters/channels involved in light-enhanced calcification.After 3 h of exposure to light, there was a transient decrease in the transcript level of T. squamosa. Giant clams are known to absorb and assimilate ammonia from the external medium during insolation [4+ into glutamine, in the ctenidium of T. squamosa, are up-regulated by light exposure. Light also enhances the transcript level and protein abundance of a Na+/H+ exchanger 3-like transporter, which mediates H+ efflux in exchange for Na+ uptake in a 1:1 stoichiometry, in the ctenidium, indicating that it may be involved in increased H+ excretion in pursuance of whole-body acid-base balance during light-enhanced calcification [2+-ATPase (PMCA) in the inner mantle of T. squamosa [T. squamosa and a novel explanation of light-enhanced calcification in general [Porites lutea and Acropora variabilis were mostly enhanced by blue light, but photosynthesis was less efficient under that part of the spectrum. They suggested that blue light photoreceptors in coral tissue could be the light sensor which activated a plasma membrane Ca2+-ATPase (PMCA) involved in light-enhanced calcification [T. squamosa, it would be rewarding to examine whether scleractinian corals would display similar light-dependent phenomena.Besides NKA, three other transporter and enzyme also display light-dependent expression in solation , 54. Recsolation  have repfication . In addisquamosa . As PMCA general . Cohen e general  reportedfication . In viewT. squamosa appear to be energy-wasteful [T. squamosa can afford energetically to regulate light-dependent processes through transcriptional and translational changes. Light can be detected by the siphonal eyes located at the surface of the host’s hypertrophied siphon [Symbiodinium which are known to possess rhodopsin [As transcription and translation are energy-dependent processes, daily changes in gene and protein expression levels in wasteful . Howeverwasteful –59. The wasteful , 61 and wasteful , 62–64. d siphon , which thodopsin . It has hodopsin , 25, 26.T. squamosa requires the supply of Ca2+ and HCO3− from the hemolymph to the extrapallial fluid through the shell-facing epithelium of the inner mantle, and the removal of H+ in the opposite direction. Many transporters and channels systems located along the apical and basolateral membranes of these epithelial cells could be involved in generating the transepithelial ion fluxes. Given that it is capable of vectorial transport, the distribution of the transporters between the apical and basolateral membranes of the shell-facing epithelium must be asymmetrical. Sano et al. reported that the shell of Tridacna derasa displayed diurnal variations of Sr/Ca ratio [2+-ATPase which would selectively incorporate Ca2+ at high-calcification rates during light-enhanced calcification, and result in widened daytime growth band with reduced Sr/Ca ratio in the giant clam shell. Besides Ca2+-ATPase, other transporters like NCX (SLC8) [+ gradient generated by the basolateral NKA. Indeed, we obtained results which confirm that the gene and protein expression levels of NCX/NCX in the inner mantle are also light-dependent. As NCX employs the Na+ gradient to transport 3 Na+ in and 1 Ca2+ out of the cell, an increase in NKA activity is required to maintain the intracellular Na+ homeostasis of these epithelial cells in the shell-facing epithelium. Hence, efforts should be made in the future to elucidate the functional roles of NCX in light-enhanced calcification in the inner mantle of T. squamosa.Shell formation in Ca ratio . Their rX (SLC8) , bicarboX (SLC8) , 67–69 aT. squamosa. Exposure to light for 12 h leads to a significantly increase in protein abundance of NKAα in the inner mantle and the basolateral NKAα-immunofluorescence along its shell-facing epithelium, indicating that NKA may play an indirect role in light-enhanced calcification. These results provide indirect evidence on the involvement of Na+-coupled transporters in light-enhanced calcification, and a firm basis for further investigation on vectorial Na+-dependent ion transport across the shell-facing epithelium of the inner mantle. It is probable that many of the transporters involved in the calcification process are expressed differentially between the apical and basolateral membranes. However, it is unlikely that every epithelial cell would express exactly the same transporters and perform identical ion transport functions. Hence, future experiments should aim to elucidate membrane-specific transport systems and different cell types involved in the vectorial transport of various ions in the shell-facing of the inner mantle in order to fully understand the mechanisms of light-enhanced calcification in T. squamosa.NKAα has a basolateral localization in the shell-facing epithelium of the inner mantle in


Zea mays ssp. mays) is 1 of 3 crops, along with rice and wheat, responsible for more than one-half of all calories consumed around the world. Increasing the yield and stress tolerance of these crops is essential to meet the growing need for food. The cost and speed of plant phenotyping are currently the largest constraints on plant breeding efforts. Datasets linking new types of high-throughput phenotyping data collected from plants to the performance of the same genotypes under agronomic conditions across a wide range of environments are essential for developing new statistical approaches and computer vision–based tools.Maize (A set of maize inbreds—primarily recently off patent lines—were phenotyped using a high-throughput platform at University of Nebraska-Lincoln. These lines have been previously subjected to high-density genotyping and scored for a core set of 13 phenotypes in field trials across 13 North American states in 2 years by the Genomes 2 Fields Consortium. A total of 485 GB of image data including RGB, hyperspectral, fluorescence, and thermal infrared photos has been released.Correlations between image-based measurements and manual measurements demonstrated the feasibility of quantifying variation in plant architecture using image data. However, naive approaches to measuring traits such as biomass can introduce nonrandom measurement errors confounded with genotype variation. Analysis of hyperspectral image data demonstrated unique signatures from stem tissue. Integrating heritable phenotypes from high-throughput phenotyping data with field data from different environments can reveal previously unknown factors that influence yield plasticity. The green revolution created a significant increase in the yields of several major crops in the 1960s and 1970s, dramatically reducing the prevalence of hunger and famine around the world, even as population growth continued. One of the major components of the green revolution was new varieties of major grain crops produced through conventional phenotypic selection with higher yield potentially. Since the green revolution, the need for food has continued to increase, and a great deal of effort in the public and private sectors is devoted to developing crop varieties with higher yield potential. However, as the low-hanging fruit for increased yield vanish, each new increase in yield requires more time and resources. Recent studies have demonstrated that yield increases may have slowed or stopped for some major grain crops in large regions of the world /[max(x)-min(x)]. False color RGB images were constructed with the values for the first principal component stored in the red channel, the second principal component in the green channel, and the third principal component in the blue channel.A consistent area of interest was defined for each zoom level to exclude the pot and nonuniform areas of the imaging chamber backdrop. Within that area, pixels with an intensity value greater than 70 in the red channel were considered to be plant pixels. The aggregate fluorescence intensity was defined as the sum of the red channel intensity values for all pixels classified as plant pixels within the region of interest, and the mean fluorescence intensity as the aggregate fluorescence intensity value divided by the number of plant pixels within the region of interest.Two methods were used to predict plant biomass. The first was a single variable model based on the number of zoom level–adjusted plant pixels identified in the 2 RGB side view images on a given day. The second was a multivariate model based upon the sum of plant pixels identified in the 2 RGB side views, sum of plant pixels identified in the 2 RGB side views plus the RGB top view, aggregate fluorescence intensity in the 2 side views, aggregate fluorescence intensity in the 2 side views plus the top view, number of plant stem pixels identified in the hyperspectral image, and number of plant leaf pixels identified in the hyperspectral image. Traits were selected to overlap with those employed by Chen et al.  where pohttp://plantvision.unl.edu/dataset under the terms of the Toronto Agreement. To validate the dataset and ensure that plants had been properly tracked through both the automated imaging system and ground truth measurements, a simple script was written to segment images into plant and not-plant pixels , fluorescence images (4.3%), and hyperspectral images (44.6%). A subset of the RGB images within this dataset were previously analyzed in Choudhury et al. , and wer.1%, fluor = 0.91). Estimates of biomass using both software packages were more correlated with each other (r = 0.96) than either was with ground truth measurements. This suggests that a significant fraction of the remaining error is the result of the expected imperfect correlation between plant size and plant mass, rather than inaccuracies in estimating plant size using individual software packages. Recent reports have suggested that estimates of biomass incorporating multiple traits extracted from image data can increase accuracy [r values) of the estimated biomass measures with ground truth data were 0.949, 0.958, 0.925, and 0.951 for multivariate linear model, MARS, Random Forest, and SVM, respectively.Based on the segmentation of the image into plant and non-plant pixels, plant height was scored as the y-axis dimension of the minimum bounding box. Plant area was scored as the total number of plant pixels observed in both side view images after correcting for the area of each pixel at each zoom employed (see the Methods). Similar approaches to estimate plant biomass have been widely employed across a range of grain crop species including rice , wheat , barley accuracy . We testThe residual value-difference between the destructively measured biomass value and the predicted biomass value based on image data and the linear regression line equation—was calculated for each individual plant Fig. C. Using r = 0.97) than the primary dataset, likely as a result of the smaller amount of genetic variability among these plants , Fig. A, false Based on this observation, an index was defined that accurately separated plant pixels into leaf and stem (see the Methods). Stem pixels were segmented from the rest of the plant using an index value derived from the difference in intensity values observed in the 1056-nm and 1151-nm hyperspectral bands. This methodology was previously described . The refIn conclusion, while the results presented above highlight some of the simplest traits that can be extracted from plant image data, these represent a small fraction of the total set of phenotypes for which image analysis algorithms currently exist, and those in turn represent a small fraction of the total set of phenotypes that can potentially be scored from image data. Software packages already exist to measure a range of plant architectural traits such as leaf length, angle, and curvature from RGB images ,37. ToolProject name: Maize Phenotype Maphttps://github.com/shanwai1234/Maize_Phenotype_MapProject home page: Operating system(s): LinuxProgramming language: Python 2.7Other requirements: OpenCV module 2.4.8, Numpy >1.5, CMake > 2.6, GCC > 4.4.x, Scipy 0.13License: BSD 3-Clause LicenseGigaScience database, GigaDB [The image data sets from 4 types of cameras, pot weight records per day, and ground truth measurements with corresponding documentation for 32 maize inbreds and same types of image data for 2 maize inbreds under 2 stress treatments were deposited in the CyVerse data commons under a CC0 license . All ima, GigaDB .Figure S1. Correlation of fresh weight biomass with total number of plant pixels identified in 2 side view images for maize plants destructively sampled at 8 different time points between 13 days and 39 DAP.DAP: days after planting; GBS: genotyping by sequencing; LED: light-emitting diode; MARS: Multivariate Adaptive Regression Splines; NDVI: Normalized Difference Vegetation Index; NIR: near-infrared; RGB: an image with separate intensity values for the red, blue and green channels; SNP: single nucleotide polymorphism; SVM: Support Vector Machines; UNL: University of Nebraska-Lincoln; PA0: Plant Area calculated from a 0-degree image ; PA90: Plant Area calculated from a 90-degree image ; PCA: principal component analysis; PH0: Plant Height calculated from a 0-degree image; PH90: Plant Height calculated from a 90-degree image; PW0: Plant Width calculated from a 0-degree image; PW90: Plant Width calculated from a 90-degree image; PF0: average of plant fluorescence intensity in 0-degree; PF90: average of plant fluorescence intensity in 90 degrees.The authors declare that they have no competing interests.This research was supported by the Nebraska Corn Board (Award #88-R-1617-03), the Iowa Corn Board, the National Science Foundation under Grant No. OIA-1557417, and Internal University of Nebraska funding to J.C.S. The sources of funding had no role in the design of the study; the collection, analysis, or interpretation of data; or in writing the manuscript.J.C.S., and Y.Q. designed the experiment; V.S., J.C.S., and Z.L. performed data acquisition; Z.L., P.P., Y.Q., Y.X., Y.G., and J.C.S. analyzed and interpreted the data; Z.L. and J.C.S. produced and curated the metadata; Z.L. and J.C.S. implemented software; Z.L. and J.C.S. prepared the initial draft. All authors reviewed the manuscript.GIGA-D-17-00187_Original-Submission.pdfClick here for additional data file.GIGA-D-17-00187_Revision-1.pdfClick here for additional data file.GIGA-D-17-00187_Revision-2.pdfClick here for additional data file.Response-to-Reviewer-Comments_Original-Submission.pdfClick here for additional data file.Response-to-Reviewer-Comments_Revision-1.pdfClick here for additional data file.Reviewer-1-Report- -- Dijun Chen20 Aug 2017 ReviewedClick here for additional data file.Reviewer-1-Report-(Revision-1) -- Dijun Chen28 Sep 2017 ReviewedClick here for additional data file.Reviewer-2-Report- -- Cory Hirsh24 Aug 2017 ReviewedClick here for additional data file.Reviewer-2-Report-(Revision-1) -- Cory Hirsh13 Oct 2017 ReviewedClick here for additional data file.Supplement FigureClick here for additional data file.


Asynchronous intrafascicular multi-electrode stimulation (aIFMS) of small independent populations of peripheral nerve motor axons can evoke selective, fatigue-resistant muscle forces. We previously developed a real-time proportional closed-loop control method for aIFMS generation of isometric muscle force and the present work extends and adapts this closed-loop controller to the more demanding task of dynamically controlling joint position in the presence of opposing joint torque. A proportional-integral-velocity controller, with integrator anti-windup strategies, was experimentally validated as a means to evoke motion about the hind-limb ankle joint of an anesthetized feline via aIFMS stimulation of fast-twitch plantar-flexor muscles. The controller was successful in evoking steps in joint position with 2.4% overshoot, 2.3-s rise time, 4.5-s settling time, and near-zero steady-state error. Controlled step responses were consistent across changes in step size, stable against external disturbances, and reliable over time. The controller was able to evoke smooth eccentric motion at joint velocities up to 8 deg./s, as well as sinusoidal trajectories with frequencies up to 0.1 Hz, with time delays less than 1.5 s. These experiments provide important insights toward creating a robust closed-loop aIFMS controller that can evoke precise fatigue-resistant motion in paralyzed individuals, despite the complexities introduced by aIFMS. Paralysis due to spinal cord injury or stroke can leave a person with intact peripheral nerves and muscles, but deficient volitional motor control, thereby reducing their health and quality of life. Many paralyzed individuals consider restoration of lost basic motor functions like grasping and walking as important behaviors that could improve their quality of life  used in this study, have enabled the selective activation of small groups of motor-units within a single targeted muscle  real-time closed-loop control method for determining aIFMS per-electrode stimulation intensities (stimulus pulse durations) to evoke precise, fatigue-resistant, isometric muscle forces in an anesthetized feline  for dynamic joint-angle feedback control that includes integrator anti-windup strategies to improve system response characteristics. PID controllers have been used to control FNS for dynamic upper-limb and lower-limb movements using single-electrode per muscle strategies, even in the presence of fatigue and system non-linearities Veltink, , though Chronic survival experiments were conducted on an adult female feline using procedures approved by the University of Utah Institutional Animal Care and Use Committee. A 100-electrode USEA  at a dosage of 10 mg/kg. The animal was then intubated and mechanically ventilated. Anesthesia was maintained with Isoflorane  at a level of 0.5–1.5%, which has been shown to suppress spinal reflexes  stimulation was delivered via USEA electrodes using a custom-built, multi-channel stimulator, using stimulus pulse durations between 0.2 and 1024 μs with 0.2-μs resolution . All presented data were postprocessed in MATLAB using a forward and backward 4th-order Butterworth low-pass filter with a 50 Hz cutoff frequency.For the initial USEA calibration  was measured for each of the six electrodes with the ankle joint-angle fixed at 90 deg., following the procedures discussed in the prior study. Because the closed-loop experiments involved non-isometric contractions, and because Tpr changes as the muscle length changes  ms when the joint-angle was 20 deg. to (Tpr −5) ms when the joint-angle was 160 deg.The control strategy used in this experiment required knowledge of the time from stimulation via each USEA electrode to the peak of the evoked twitch-torque response  controlled by a microcontroller , schematically shown in Figure The stimulus pulse duration that was just below the torque producing threshold, along with the lowest stimulus pulse duration that evoked the maximal twitch-torque, were determined from twitch-torque recruitment curves measured for each electrode. These were set as the minimum and maximum allowable stimulus pulse durations for each electrode, creating a bounded input system , θe, j is the joint-angle position, ge is an additional gain factor based on experimentally determined recruitment-curve slopes for each electrode, determined by Equation (2), and Δt is the time step between per-electrode stimulations (100 ms). The limit of summation, n, was initially set to j−1, which allows for complete integration across all error samples. Adjustments to this limit, which provided closed-loop response improvements, will be described in the following sections.The real-time closed-loop aIFMS control strategy and system was previously created for evoking desired isometric forces , 2w + 1 is the length of the sampling window over which the error function is evaluated, where w was set to 5 ms of samples in all experiments, and Tpr is the predicted time of peak twitch-torque for the e-th electrode during the j-th cycle of stimulation . The joint-angle position in Equation (1), θe, j, was determined as the mean of θm, determined over the same sampling window used in Equation (3).Similarly to what was done for force-feedback control , the velocity gain was slowly increased until the system was near critically damped, which reduced the transient overshoot and decreased the settling time. The velocity term in Equation (1) is made negative because the damping is created by opposing the speed of the motion; thus, increased velocity results in decreased stimulus intensity. Finally, using proportional-plus-velocity-plus-integral control (PIV), the integrator gain was slowly increased until the overshoot reached 10%.In these experiments, the integrator was used to drive the steady-state error toward zero for desired step motions. Although the integrator works well at reducing steady state error, it can also cause large overshoot due to integrator windup. Integrator windup often occurs when there is a large and rapid change in a desired response, e.g., during a stepped change in the desired response, and the integral of the error accumulates substantially during the rising phase of the closed-loop system response, often causing large overshoot that is not released until the integrator is unwound by error in the opposite direction. In our system, additional integrator windup occurred because of the inherent delay in the system due to the controller looking one full cycle backward in time, and because the initial stimulus intensity was pre-threshold, requiring time for the controller to increase the stimulus intensity high enough to overcome the oppositional loading torque.N most recently acquired error values were summed; n = N−1 in Equation (1). The value of N was empirically determined to be eight error samples (800 ms) because this created a critically-damped response. Also, because the reduction in joint position was based solely on relaxation of muscle fibers against the joint loading torque, the integrator tended to cause an oscillatory response near a stationary desired position, which was reduced by removing the integrator for error values less than zero, i.e., when the evoked response was greater than the desired response. These improvements were implemented by modifying the control law of Equation (1) so that the limit of summation, n, was set to 7, and by setting ki = 0 when Ee, j < 0.To reduce the integrator windup, the integrator was made leaky, in effect turning the integrator into a lag compensator Nise, . This wa%OS) was measured as the percent difference between the peak evoked position and the mean evoked position during the last half-second of stimulation (steady-state position). Rise time (Tr) was measured as the time from step onset to when the system reached 90% of the steady-state position. Time-to-peak (Tp) was measured as the time from the step onset to when the system reached the peak evoked position. Settling time (Ts) was measured as the time from the step onset to when the system settled to within ±2% of the steady-state position. Steady-state error (SSE) was measured as the difference between the steady-state position and the desired step position.Various step sizes from 15 to 75 deg. were then tested along with various loading torques ranging from 0.2 to 0.8 Nm, using the tuned PIV closed-loop controller gains and the adapted control law of Equation (1) as described above. For these desired step responses, the closed-loop system was experimentally evaluated for the overall evoked joint position percent overshoot, rise time, time-to-peak, settling time, and steady-state error. Percent overshoot (Td) of the closed-loop system. Amplitude error (Ea) was also used as a performance metric and was determined as the per-sample difference between the time-shifted evoked response and the desired joint position , phase delay (Φd), and peak-to-peak response amplitude for 0.05–0.4 Hz trajectories, which are within the bounds of normal periodic human lower limb movements that occur at speeds up to a maximum of 2.0 Hz  closed-loop system responses for several values of the proportional gain, kv, are shown in Figure kp held constant at 8.0 μs/deg. The velocity gain was tuned until the system was nearly critically damped, yielding a response with Tr = 2.95 s, Tp = 6.13 s, %OS = 0.33%, and SSE = 0.63% or 0.19 deg.Velocity control was then added to the controller (PV control) to reduce the transient overshoot and oscillatory behavior caused by the large proportional gain. Representative PV closed-loop system responses for various values of the velocity gain, ki, are presented in Figure kp held constant at 8.0 μs/deg. and kv held constant at 10 μs/(deg./s). The integrator gain was tuned to produce a faster system response, Tr = 1.55 s, with transient overshoot of 11.38%. Integral control is not commonly used to speed up the transient response, though it had that effect in these experiments due to system delays leading to large integrator windup. To reduce settling time, the integrator was turned off when the error was less than zero (descending motion). This resulted in the settling time decreasing from 6.98 s to 6.02 s without affecting any other response metrics. The integrator was then made leaky to deal with integrator windup, i.e., only the 8 most recently acquired error values were numerically integrated, and only when the error was greater than zero.Integral control was then added to the controller (PIV control) to ensure that the steady-state error stays near zero. Representative PIV closed-loop system responses for increasing values of the integrator gain, Tr = 2.28 s, Tp = 4.22 s, Ts = 4.53 s, %OS = 2.39%, and <1% SSE, using the following controller gains: kp = 8.0 μs/deg., kv = 10.0 μs/(deg./s), ki = 5.0 μs/(deg.·s). Table A representative tuned PIV closed-loop system response is demonstrated in Figure Using the tuned PIV controller gains, the robustness of the controlled response to various step sizes and loading torques was tested along with the ability of the controller to reject disturbance torques. In addition, the reliability of the controlled response over experimental sessions was tested using the same six electrodes and controller gains.Tr = 2.31 s, Tp = 4.68 s, Ts = 8.41 s, and had more overshoot, %OS = 15.6%. The PIV controlled response for several desired step sizes against a loading torque of 0.4 Nm, and with a starting position of 25 deg., is presented in Figure The PIV controlled response for a 30-deg. desired step with increasing loading torques is displayed in Figure t = 16 s. The increased load was maintained for 15 s and then the loading torque was decreased from 0.6 to 0.4 Nm. When the disturbance load was initially applied, the system settled in 1.88 s with near-zero overshoot, and when the disturbance load was removed, the system settled in 5.56 s, also with near-zero overshoot.A representative closed-loop controlled response to an applied disturbance torque is demonstrated in Figure Tr = 2.07 ± 0.19 s, Tp = 4.13 ± 0.16 s, Ts = 6.68 ± 0.45 s, %OS = 3.65 ± 1.55%. The PIV controller was tested to the limit where activated muscle fibers showed strong fatigue, as presented in Figure Using the same six electrodes and PIV controller gains, representative closed-loop controlled responses over time are shown in Figure Ea = 0.21 ± 1.02 deg., Td = 1.25 s for the ramp-up phase and Ea = −0.24 ± 0.98 deg., Td = 1.45 s for the ramp-down phase. The time delay remained at approximately 1.3 s when the speed of the desired ramp trajectories increased. However, the error of the controlled response increased and the smoothness of the evoked motion decreased, as shown in Table The closed-loop PIV controller was able to evoke ramped joint-angle trajectories with slopes ranging from 2 to 64 deg./s against a 0.4-Nm loading torque, as presented in Table Td = 1.46 s and phase delay of Φd = 26.3 deg., as shown in Figure The closed-loop PIV controller was also able to evoke sinusoidal joint-angle trajectories with frequencies ranging from 0.05 to 0.4 Hz against a 0.4-Nm loading torque, as displayed in Table Using the same six electrodes and PIV controller gains, the closed-loop controlled response for the time-varying trajectories remained consistent over time, as shown in Figures The results presented in this paper demonstrate that tracking of desired step, ramp, and sinusoidal joint-angle trajectories can be successfully achieved with real-time joint-angle feedback control of aIFMS. This demonstration is important because although prior experiments evoked precise time-varying isometric muscle force trajectories , and the maximum evoked torque remained strong (14–20 Ncm for session one and 20–25 Ncm for session two). A further experimental session was desired around 30 days from the original, but the implant was no longer viable to a degree where stimulation via these electrodes produced strong muscular output (maximum evoked torques were below 6 Ncm). These are important outcomes of these experiments that extend the successful chronic use of USEAs for stimulation and recording, while providing insights toward limitations (Branner et al., This paper demonstrated the first successful closed-loop dynamic limb position control using aIFMS delivered via a chronically implanted USEA. The approach presented in the paper, and experiments validating it, represent an important step toward creating the next generation of clinically-viable, fatigue-resistant, controlled FNS-based neuroprostheses to aid and restore lost motor function in persons with paralysis.MF was a graduate student during this research. He performed all the experiments and is the main author of this manuscript. The other four authors, VM, GC, RN, and SM, all contributed to the experimental design of this research, participated during experiments, and assisted in creation of this manuscript.This work was supported in part by USAMRMC W81XWH-10-1-0931, University of Utah, and Oregon State University.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aims. The prognostic role of the proteases uPA and PAI-1, as well as tumor budding, in colon cancer, has been investigated previously. Methods. We provide 6-year follow-up data and results of the validation set. The initial test set and validation set consisted of 55 colon cancers and 68 colorectal cancers, respectively. Tissue samples were analyzed for uPA and PAI-1 using a commercially available Enzyme-Linked Immunosorbent Assay (ELISA). Tumor budding was analyzed on cytokeratin-stained slides. Survival analyses were performed using cut-offs that were determined previously. Results. uPA was not prognostic for outcome. PAI-1 showed a trend towards reduced cancer specific survival in PAI-1 high-grade cases . The combination of high-grade PAI-1 and tumor budding was associated with significantly reduced cancer specific survival . After pooling the data from both sets, multivariate analyses revealed that the factors pN-stage, V-stage, and a combination of tumor budding and PAI-1 were independently prognostic for the association with distant metastases. Conclusions. A synergistic adverse effect of PAI-1 and tumor budding in uni- and multivariable analyses was found. PAI-1 could serve as a target for anticancer therapy. The role of the microenvironment in cancer has recently gained growing attention. Different conditions can inhibit or enhance tumor growth and dissemination. For example, tumor-associated inflammatory reactions can induce tumor inhibition or promotion. Bioactive molecules released from inflammatory cells to the microenvironment support different hallmark capabilities, including proliferation, cell death limitation, angiogenesis, and matrix modification . BecauseThe test set has been described before. In brief, it consists of 59 colon specimens with 55 cancer lesions that were collected between August 2007 and September 2008. Follow-up data were provided by the clinical and population-based cancer registry of Augsburg, Augsburg, Germany. Additional information was retrieved from the files of the Institute of Pathology and the clinic information system. The physicians that were responsible for further care were contacted to gain information, especially concerning disease control or progression. This study was approved by the ethical committee of the Landesärztekammer Bayern. Informed and written consent was obtained from all patients.The 68 colorectal cancer cases of the validation set were collected between April and November 2014. This study was approved by the internal review board of the Klinikum Augsburg on the basis of the recommendation by the ethical committee of Landesärztekammer Bayern regarding the previous study. The inclusion criteria for this study were established or suspected colorectal cancer and an elective oncological resection. Emergency surgery or noncurative intentions were criteria for exclusion.3 tissue block was cut from the invasive tumor region in the fresh tissue immediately after receiving the unfixed specimen. The time between resection and sample collection was less than 30 minutes. In 13 cases from the test set, samples from nonneoplastic regions were also collected. The samples were stored at −20°C for a maximum of 14 days before performing ELISA. Malignancy and the estimation of proportion of the neoplastic cells in 10% steps were confirmed by H&E histology in all samples for the ELISA.The specimen preparation has been described before. In brief, a 1 cm2SO4 and the absorption of the solution was measured using a microwell reader at 450 nm. The levels of uPA and PAI-1 were expressed in nanograms per milligram (ng/mg) of tumor protein. Based on the evaluation of the test set [A commercially available ELISA kit for uPA and PAI-1 that is certified for usage in breast cancer was used (Femtelle Test (EF 899), Sekisui Diagnostics, Stamford, CT). The test was conducted according to the protocol recommended by the manufacturer and has been described before . In brieTumor budding was defined as isolated tumor cells or clusters of up to four cells at the invasion front according to Ueno et al. Because of the usage of a microscope with an eyepiece with a field number of 25, the cut-off for high-grade budding was adjusted to ≥30 buds/20-fold magnification in comparison to the original publication . The evaImmunohistochemistry for the mismatch repair proteins PMS2 and MSH6 was performed to determine the mismatch repair status in the cases of the validation set where evaluations concerning microsatellite instability (MSI) and/or mismatch repair deficiency (MMRd) have not been performed in the frame of the routine diagnostic. The following diagnostic antibodies were used: PMS2  . All reactions were developed using the Ventana Ultravision detection system .χ2 test or Fisher's Exact test depending on the number of observations. Backward stepwise logistic regression analysis was used to identify independent predictors of metastatic disease. For the survival analysis, Kaplan-Meier Curves were calculated and differences were analyzed with the Log-Rank test. The mean survival times were calculated because the median survival was not reached in most analyses. For the determination of the median follow-up time, the method of Schemper and Smith was used. The Cox regression proportional hazards model was used for the multivariate analysis of cancer specific analysis. The Cohens-Kappa value was calculated to evaluate the interobserver variability during the tumor budding evaluation. All calculations were performed using the Sigma Plot 13.0 software package . P values < 0.05 were considered significant.The Mann–Whitney Rank Sum test was used to compare numeric values. Correlations were calculated with Pearson Product Correlation. Tabulated data were compared using the The test set consists of 55 colon cancer cases. Forty-seven of these cases met the criteria for survival analysis. The basic clinicopathological data have been described before and are briefly given in P = 0.735), respectively. However, a clear trend towards adverse outcome was found for PAI-1. Cases with high PAI-1 levels showed a mean CSS time of 68 months (95% CI: 47–89) compared to low PAI-1 level cases with a CSS time of 83 months (95% CI: 74–93) (P = 0.091). High-grade tumor budding was also associated with impaired survival, with a mean CSS time of 71 months (95% CI: 53–89) versus 83 months for low-grade tumor budding (95% CI: 73–33) (P = 0.187). Although this difference was not statistically significant, the Kaplan-Meier curves showed a clear discrimination between the two groups. After combining the parameters tumor budding and PAI-1, the cases with no or only a single positive revealed a significantly higher CCS in comparison to cases that were positive for both. The mean times for negative/single positive versus double positive cases were 83 months (95% CI: 74–91) and 60 months (95% CI: 29–91), respectively (P = 0.021). The Kaplan-Meier curves are shown in  N-stage, vascular invasion, PAI-1, tumor budding, and the combination of PAI1 and tumor budding, only N-stage was found to be independently prognostic. However, the combination of PAI-1 and tumor budding just failed independence, with a P-for-Enter of 0.057.Cancer specific survival was not different in cases with low and high uPA levels with a mean CSS time of 80 months (95% CI: 70–91) versus 76 months (95% CI: 59–94) .All samples contained neoplastic cells. The mean percentage of the vital tumor was 640.207 ± 22% (Range 5–90%). There was a weak nonsignificant association between the tumor amount and the level of uPA and no association with PAI-1  with each other. However, in this set, tumor budding did not correlate significantly with uPA or PAI-1 (R = 0.08 and R = 0.218) (The mean uPA and PAI-1 levels were 4.0 ± 2.2 and 38.1 ± 33.7 ng/mg protein. Both parameters showed a strong correlation (= 0.218) .P = 0.043) . Further significant differences were found for uPA and PAI-1 between the different pT-stages     . The ass Figures  and grad Figures . Additio= 0.153) , whereas= 0.153) .P = 0.07) and a significantly higher rate of high-grade tumors in the uPA-high group . Both parameters were also significantly differentially distributed in the PAI-1 low versus high groups .The clinicopathological data, stratified according to the cut-off values for uPA and PAI-1 that were determined during the first evaluation of the test set, are given along with the data for tumor budding in P = 0.05) and node positivity . It was also significantly associated with a left side location , venous invasion , and metastatic disease .High-grade tumor budding was more highly associated with male gender (P = 0.051 (OR: 3.5). The multivariate analyses revealed that the factors pN-stage, venous invasion, and combination of tumor budding and PAI-1 were independently prognostic for the occurrence of distant metastases.After pooling the data from the test and the validation set, the risk for the occurrence of distant metastases was calculated for pT-stage, pN-stage, venous invasion, grading, uPA, PAI-1, tumor budding, and the combination of tumor budding and PAI-1 . Signifiβ-catenin upregulates uPA expression. The evaluation of our test set revealed strong correlations between tumor budding and uPA and PAI-1 levels [P = 0.167) in this direction.Here, we provide follow-up data from a prospective study that was initially performed in 2007/2008 to investigate the role of the serine proteases uPA and PAI-1 in tumor budding in colon cancer . Moreove1 levels . HoweverIn our initial study, we showed an association between the tissue levels of uPA and PAI-1 and aggressive histopathological features of colon cancers. The recently finished validation study confirmed the role of these proteases as negative prognostic markers in colon cancers. This was true when the mean values were compared and also when the collection was stratified according to previously determined cut-off values. However, it has to be stated that the combinations of factors that were associated with each other were not completely identical , which iA multivariable analysis performed after pooling the data from both sets revealed that pN-stage, V-stage, tumor budding, and PAI-1 were independently associated with distant metastases. The prognostic role of PAI-1 in colorectal cancers has been evaluated by several other groups, and the overwhelming majority of studies confirmed that PAI-1 is an adverse factor . An overIn the analyses of the test and validation sets, UPA was found to be inferior concerning its prognostic significance in comparison to PAI-1 and tumor budding. Kushlinskii et al. also found PAI-1, but not uPA, to be prognostic in colorectal cancer . Howeverκ-value of 0.48. It is astonishing that, despite these limitations, tumor budding has been demonstrated to be a robust adverse factor many times. An increased risk for lymph node metastases in pT-1 cancers has been confirmed in systematic reviews and meta-analyses [Tumor budding is gaining recognition as a promising and robust prognostic factor in colorectal cancer. It is especially interesting for the evaluation of malignant polyps in the preoperative setting and in stage II cancers . Its proanalyses –36. Seveanalyses –40, whicDespite investigating another set of 68 cases in addition to the initial collection, this study is still hampered by a relatively low case number. Nevertheless, the data of this study with a prospective design can serve as a basis for further investigations. We found a synergistic adverse effect of PAI-1 and tumor budding in uni- and multivariable analyses. This supports the thesis that stromal degradation facilitates the migration of detached tumor cells. Because of the association with the histological type further investigations should focus on this by comparing collectives with identical UICC stages, MSI status, and/or locations. Because PAI-1 inhibitors are already available, additional investigations should address the question of whether PAI-1 could serve as a target for anticancer therapy .